The biochemical mechanism of fatty acid biosynthesis is universally similar among all organisms. Generally, fatty acids are synthesized by the repeated iteration of four reactions, which start with an acyl-primer, which is elongated, two carbons per cycle, using carbon atoms derived from a malonyl moiety. The four sequential reactions that make up this cycle generate 3-ketoacyl-thioester, 3-hydroxyacyl-thioester, and 2-enoyl-thioester derivative intermediates, and finally an acyl-thioester derivative that is two carbons longer than the initial acyl primer. In bacteria, typified by the Escherichia coli system, and higher plant plastids, these reactions are catalyzed by a dissociable, type II fatty acid synthase that is composed of the four enzymes 3-ketoacyl-ACP synthase (KAS), 3-ketoacyl-ACP reductase (encoded by fabG), 3-hydroxyacyl-ACP dehydratase (encoded by fabA), and enoyl-ACP reductase (encoded by fabI) (Rock et al., Biochim. Biophys. Acta 1302: 1-16 (1996)). In contrast, a type I fatty acid synthase, which is composed of four enzyme components that occur as domains on a multifunctional protein(s), occurs in other eukaryotes (Jenni et al., Science 311: 1263-1267 (2006); and Maier et al., Science 311: 1258-1262 (2006)). However, in both type I and type II fatty acid synthase systems acyl derivatives are bound to phosphopantetheine cofactors.
In the type II fatty acid synthase system, there are three genetically and biochemically distinct KAS isomers, namely KASI (encoded by fabB), KASII (encoded by fabF), and KASIII (encoded by fabH) (Rock et al. (1996), supra; Garwin et al., J. Biol. Chem. 255: 11949-11956 (1980)); and Jackowski et al., J. Biol. Chem. 262: 7927-7931 (1987)). Their functions have been studied extensively in E. coli. They differ in their specificities for acyl-thioester substrates, having optimum activities for substrates of different acyl-chain lengths and different thioesters. While KASI and KASII catalyze the condensation between acyl-ACP (of longer acyl-chain length) with malonyl-ACP substrates, KASIII specifically utilizes acetyl-CoA as a substrate for the condensing reaction with malonyl-ACP (Tsay et al., J. Biol. Chem. 267: 6807-6814 (1992); and Heath et al., J. Biol. Chem. 271: 1833-1836 (1996)), and thus initiates fatty acid biosynthesis.
The general mechanism of fatty acid biosynthesis in Gram-positive bacteria, such as Bacillus subtilis, is similar to that of E. coli (Magnuson et al., Microbiol. Rev. 57: 522-542 (1993)). One major difference is that B. subtilis produces large quantities of branched-chain fatty acids (BCFAs) and unsaturated fatty acids as a result of the expression of a unique Δ5 desaturase (Aguilar et al., J. Bacteriol. 180: 2194-2200 (1998)). The BCFAs and the unsaturated fatty acids together maintain membrane fluidity in response to lower growth temperatures. The BCFAs are branched with methyl groups at the iso- and anteiso positions (i.e., 13-methyltetradecanoic, 12-methyltetradecanoic acid, and 14-methylpentadecanoic acid), and they are biosynthesized by a type II FAS that has the ability to initiate this process by using branched acyl-CoAs that are derived from the branched chain amino acids, leucine, isoleucine, and valine (Willecke et al., J. Biol. Chem. 246: 5264-5272 (1971)). Thus, the B. subtilis FAS enzyme must have the capacity to utilize such branched acyl-CoA substrates. Genomics-based analysis of the B. subtilis genome has led to the identification of KASII (Shujman et al., J. Bacteriol. 183: 3032-3040 (2001)) and KASIII homologous genes; however, it appears that this bacterium does not contain a sequence-recognizable KASI homolog. In B. subtilis KASII is an essential enzyme, which is encoded by yjaY. Two B. subtilis KASIII-encoding genes, bfabHA (yjaX) and bfabHB (yhfB), have been characterized, and these have the capacity to catalyze the condensation of branched acyl-CoAs with malonyl-ACP (Choi et al., J. Bacteriol. 182: 365-370 (2000); and Smirnova et al., J. Bacteriol. 183: 2335-2342 (2001)). These two genes code for 312- and 325-residue proteins that share 43% sequence identity.
KASIII has been characterized in several bacterial (Tsay et al., J. Biol. Chem. 267: 6807-6814 (1992); Han et al., J. Bacteriol. 180: 4481-4486 (1998); Qiu et al., J. Biol. Chem. 274: 36465-36471 (1999); Choi et al., J. Bacteriol. 182: 365-370 (2000a); Choi et al., J. Bacteriol. 182: 365-370 (2000b); Choi et al., J. Biol. Chem. 275: 28201-28207 (2000c); Davies et al., Structure 8: 185-195 (2000); Khandekar et al., Biochem. Biophys. Res. Comm. 270: 100-107 (2000); Khandekar et al., J. Biol. Chem. 276: 30024-30030 (2001); Qiu et al., J. Mol. Biol. 307: 341-356 (2001); Revill et al., J. Bacteriol. 183: 3526-3530 (2001); Huynh et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Comm. 65: 460-462 (2009); Wen et al., Protein Expr. Purif. 65: 83-91 (2009); Singh et al., FEMS Microbiol. Lett. 301: 188-192 (2009); Gajiwala et al., FEBS Lett. 583: 2939-2946 (2009); and Pereira et al., Acta Crystallogr. D. Biol. Crystallogr. 68: 1320-1328 (2012)), protozoan (Waters et al., Mol. Biochem. Parsitol. 123: 85-94 (2002); and Prigge et al., Biochem. 42: 1160-1169 (2003)), and plant species (Clough et al., J. Biol. Chem. 267: 20992-20998 (1992); Jaworski et al., Plant Physiol. 90: 41-44 (1989); Tai et al., Plant Physiol. 106: 801-802 (1994); Abbadi et al., Biochem. J. 345 (Pt. 1): 153-160 (2000); Dehesh et al., Plant Physiol. 125: 1103-1114 (2001); Li et al., Tree Physiol. 28: 921-927 (2008); and Gonzalez-Mellado et al., Planta 231: 1277-1289 (2010)). Functionally characterized KASIII enzymes exhibit diverse substrate specificities, utilizing acyl-CoA substrates ranging from short, straight-chain acyl-CoAs (e.g. acetyl-CoA, propionyl-CoA (Choi et al. (2000a), supra; Clough et al. (1992), supra; and Abbadi et al. (2000), supra), branched-chain acyl-CoAs (e.g., isobutyryl-CoA and ante-isovaleryl-CoA (Han et al. (1998), supra; Choi et al. (2000a), supra; Khandekar et al. (2001), supra; Singh et al. (2009), supra; and Qiu et al., Protein Sci. 14: 2087-2094 (2005)) to long-chain acyl-CoAs (e.g., lauroyl-CoA, palmitoyl-CoA (Choi et al. (2000b), supra; and Scarsdale et al., J. Biol. Chem. 276: 20516-20522 (2001)).
By virtue of diverse substrate specificities shown by KASIII enzymes from different organisms, this enzyme is thought to determine the fatty acid profile of the organism, particularly the structure of the omega-end of the fatty acid products (Choi et al. (2000a), supra; Gajiwala et al. (2009), supra; and Pereira et al. (2012), supra). For example, in many Gram-positive bacteria (i.e., Bacillus subtilis, Streptomyces glaucescens, and Staphylococcus aureus), KASIII can utilize both branched-chain and straight-chain substrates, resulting in the production of both branched- and straight-chain fatty acids (Han et al. (1998), supra; Choi et al. (2000a), supra; Pereira et al. (2012), supra; and Qiu et al. (2005), supra). In contrast, KASIII from Gram-negative bacteria (e.g., E. coli) appears to prefer straight-chain acyl-CoA substrates, which results in the production of straight-chain fatty acids (Choi et al. (2000a), supra).
The active site residues and substrate binding pocket are well conserved among KASIII from different species. Three residues, Cys112, His244 and Asn274, form the catalytic triad in E. coli KASIII and carry out two half reactions as a part of the Claisen condensation (FIG. 1) of acyl-CoA and malonyl-ACP (Heath et al., Nat. Prod. Rep. 19: 581-596 (2002)). Qiu et al. (J. Biol. Chem. 274: 36465-36471 (1999)) and Davies et al. (Structure 8: 185-195 (2000)) have proposed different mechanisms for the reaction at the active site. The mechanism proposed by Davies et al., which is supported by crystallographic data, is shown in FIG. 2. The first step is the transfer of the acyl group from the acyl-CoA primer to the enzyme and covalent linkage of the acyl group to the Cys112 residue. As per Davies et al., the thiol group of Cys112 is deprotonated by the dipole effect of the α-helix in which it is located. The resulting nucleophilic thiolate ion on Cys112 attacks acyl-CoA and forms a thioester with the acyl group, with the release of CoA-SH. The second step is the entry of ionized malonyl-ACP into the active site, where it is decarboxylated through the aid of Phe205.
After decarboxylation, the resulting negative charge on its thioester carbonyl is stabilized by His244 and Asn274. A carbanion is formed on its α-carbon that attacks the acetate bound to Cys112. The tetrahedral transition state is stabilized by an oxyanion hole formed by Cys112 and Gly306, which eventually breaks down to give acetoacetyl-ACP as the product.
The US imports almost 10 million barrels of petroleum a day (U.S.E.I. Administration Monthly Energy Review (2011), www.eia.gov/energy_in_brief/foreign_oil_dependence.cfm) to create a multi-billion dollar plastics and specialty chemical industry that obtains its monomers from petroleum feedstocks. Currently, only a limited number of bio-based products are available in the market including polylactic acid (PLA), polyhydroxybutyrate (PHB), and polyethylene terephthalate (PET), which is based on 1,3-propanediol, and emerging products based on succinic acid and adipic acid (Frost and Sullivan, Global Bio-Based Plastics Market (2009), www.frost.com/prod/servlet/report-toc.pag?repid=M4A1-01-00-00-00). However, the global marketplace for the bio-plastics “green” market is projected to expand to over a billion dollars (Ceresana Research, Market Study: Bioplastics (2011), www.ceresana.com/en/market-studies/plastics/bioplastics), and with increasing awareness about reduced environmental impacts of bio-based plastics, the market for these products will continue to grow.
Additionally, the last 50 years have seen an increasing concern about climate change and increasing volatility in the price of petroleum feedstocks, which has prompted a shift toward exploring sustainable sources of chemicals and fuels. Fatty acids and their derivatives are chemically the most similar biological molecules to petroleum hydrocarbons, and are therefore the most readily reachable targets for usurping as sustainable replacements for petroleum-derived fuels and chemicals (Steen et al., Nature 463: 559-562 (2010); Handke et al., Metab. Eng. 13: 28-37 (2011); and Metzger et al., Appl. Microbiol. Biotech. 71: 13-22 (2006)). Indeed, considerable research efforts have been expended to identify the enzymology and genetic elements that are responsible for the diversity of chemical structures that can be accessed via the metabolic processes of fatty acid metabolism. Much of this success has been facilitated by the modular nature of the enzymatic machinery that underlies the process of fatty acid synthesis (FAS) and the more general polyketide biosynthesis machinery (Stewart et al., Curr. Opin. Plant Biol. 16: 365-372 (2013)). These processes iteratively condense 2-carbon precursors, but FAS follows each condensation reaction by a 3-reaction process (reduction-dehydration-reduction) that generates a fully reduced alkyl chain. Analogous to the more general polyketide synthesis mechanisms, prokaryotic FAS systems sometimes skip the final reduction reaction prior to the next condensation iteration, and thus leave a carbon-carbon double bond in the alkyl chain. In contrast to this prokaryotic anaerobic process, most eukaryotic organisms assemble the fully reduced alkyl chain and subsequently oxidize the fatty acid by aerobic reactions catalyzed by desaturases that introduce carbon-carbon double bonds or a series of homologous enzymes that can introduce oxygen into the alkyl chain to produce, for example, hydroxy- or epoxy-fatty acids. Most of these functional groups occur in relatively central positions of the alkyl chain (e.g., between the 5th and the 15th carbons of an 18-carbon fatty acid). Such modified unsaturated or oxygenated fatty acids are targets for subsequent non-biological chemical conversions that can provide access to even larger numbers of chemicals with many wide-ranging applications, such as lubricants, surfactants and polymers (Metzger, Eur. J. Lipid Sci. & Tech. 111: 865-876 (2009)).
Steen et al. ((2010), supra) reports engineering E. coli to produce fatty esters, fatty alcohols, and waxes from glucose. Free fatty acid and acyl-CoA production reportedly was improved by eliminating fatty acid degradation by knockout of the fadE gene, which is responsible for β-oxidation, and overexpression of thioesterases (TE) and acyl-CoA ligases (ACL). Overexpression of fatty acyl-CoA reductases (FAR) reportedly resulted in the production of fatty alcohols from acyl-CoA. Expression of an acyltransferase (AT) in conjunction with pdc and adhB (an alcohol forming pathway) reportedly resulted in the production of wax esters.
The formation of new carbon-carbon bonds by the condensation of an acyl-CoA substrate with the acetyl-moiety of a malonyl-thioester substrate (i.e., malonyl CoA or malonyl-ACP) (Heath et al., Nat. Prod. Rep. 19: 581-596 (2002)) by KASIII forms the basis for a diverse set of natural products that can be sub-classified as different types of polyketides. Specifically, the diketide thioester that is formed by a single KASIII-type condensation reaction can undergo additional iterations of condensation reactions, sequentially giving rise to triketides, tetraketides, pentaketides, etc. Alternatively, the diketide can undergo sequential reduction-dehydration-reduction reactions to generate an acyl-chain that is fully reduced, and two carbons longer than the initial substrate, i.e., fatty acid biosynthesis. Then again, certain metabolic processes alternate the condensation reactions with the first and second of the sequential reduction-dehydration-reduction reactions to produce hydroxylated or unsaturated natural products. An additional diversity of biochemical products can be generated by the fact that the KASIII-type enzymes utilize different acyl-CoA substrates. For example, a KASIII enzyme that uses acetyl-CoA as a substrate is used by Type II fatty acid synthase and generates the “normal” chain fatty acids, but KASIII enzymes that use branched-chain acyl-CoA substrates can be used to generate branched chain fatty acids (Choi et al., J. Bacteriol. 182: 365-370 (2000a); Gajiwala et al., FEBS Lett. 583: 2939-2946 (2009); and Pereira et al., Acta Crystallogr. D. Biol. Crystallogr. 64: 1320-1328 (2012)). Another class of KASIII-type enzymes utilizes aromatic acyl-CoAs to generate phenylpropanoid natural products, such as flavonoids, anthocyanins and stilbenes. Alicyclobacillus acidocaldarius makes 59% ω-alicyclic fatty acids naturally, primarily ω-cyclohexyl-C17:0 and -C19:0 acids (Ratledge et al., Microbial Lipids, Vol I, Academic Press, UK (1988)), and can also make ω-cyclobutyl-, ω-cyclopentyl-, and ω-cycloheptyl-acids if provided with cyclobutyl-, cyclopentyl- and cycloheptyl-acetic acids as precursors (De Rosa et al., Phytochem. 13: 905-910 (1973)). It has also been demonstrated that ω-cyclic fatty acids accumulate in a B. subtilis strain that was fed precursor ω-cyclic carboxylic acids (e.g., cyclobutanecarboxylic acid and cyclohexanecarboxylic acid) (Dreher et al., J. Bacteriol. 127: 1136-1140 (1976)). This clearly suggests that both aaKASIII and bsKASIIIb have large substrate pockets and are capable of utilizing ω-cyclic substrates, therefore resulting in corresponding ω-cyclic fatty acids. Although many KASIII structures are available (Davies et al., Structure 8: 185-195 (2000); Qiu et al., J. Biol. Chem. 275: 36465-36471 (1999); and Qiu et al., J. Mol. Biol. 307: 341-356 (2001)), the structure-function relationship that determines the substrate specificity of KASIII remains to be defined. In various attempts to understand the underlying structural basis of KASIII substrate diversity (Gajiwala et al. (2009), supra; and Pereira et al. (2012), supra), structural and sequence information has been mined, and several structural motifs and residues have been proposed to govern KASIII substrate specificity. For example, a recent study identified 22 residues that form the large CoA binding tunnel and, therefore, may have a role in defining KASIII substrate specificity (Gajiwala et al. (2009), supra).
Most known KASIII enzymes use unsubstituted, relatively inert acyl-CoA substrates, which define the chemical nature of the omega-end (ω-end) of a fatty acid; because most KASIII enzymes, including E. coli KASIII, use acetyl-CoA as the substrate in this reaction, the ω-end of the final product is an unreactive methyl group, for example (Choi et al. (2000), supra). However, KASIII from some bacteria, such as Bacillus subtilis and Staphylococcus aureus, has been shown to utilize substituted acyl-CoAs (i.e., acyl-CoAs with methyl branches at the ω-1 and ω-2 positions, e.g., isobutyryl-CoA and ante-isovaleryl-CoA) resulting in fatty acids with methyl branches at the ω-ends (Choi et al. (2000), supra; and Gajiwala et al. (2009), supra). As ω-functionalized fatty acids widen the scope of possible subsequent chemical transformations, and enable the synthesis of new building blocks for polymers, resins, films, coatings, bilayers, and micelles (Metzger et al. (2006), supra, and Zerkowski et al., J. Amer. Oil Chem. Soc. 89: 1325-1332 (2012)), such molecules are highly desirable as feedstocks in the chemical industry (Metzger et al. (2009), supra; Zerkowski et al. (2012), supra).
Of particular interest are ω and ω-1 hydroxy fatty acids as these are proposed to be excellent monomers for synthesizing polyethylene-like bio-based plastics (Lu et al., J. Am. Chem. Soc. 132: 15451-15455 (2010); and Ceccorulli et al., Biomacromolecules 6: 902-907 (2005)), and can be readily converted to macrocylic lactones (Antczak et al., Enzyme & Microbial Tech. 13: 589-593 (1991)) that have applications in the pharmaceutical industry (Omura, Macrolide Antibiotics: Chemistry, Biology and Practice, 2nd ed., Academic Press (2002)) and the flavors and fragrances industry (Theimer, Frangrance Chemistry: The science of the sense of smell, Academic Press (1982); and Vandamme et al., J. Chem. Tech. & Biotech. 77: 1323-1332 (2002)). The presence of ω and ω-1 hydroxy fatty acids in naturally occurring sophorolipids (Gorin et al., Canadian J. Chem. 39: 846-855 (1961); and Asmer et al., J. Amer. Oil Chem. Soc. 65: 1460-1466 (1988)) imparts superior functional properties to the sophorolipids as biosurfactants (Ashby et al., Biotech. Lett. 30: 1093-1100 (2008)). A wide range of possible chemical transformations to ω-1 hydroxy fatty acids has been experimentally described to result in products with enhanced functionalities (Zerkowski et al. (2012), supra).
Naturally, ω and ω-1 hydroxy fatty acids occur in glycolipids, namely sophorolipids that are synthesized by fermentation of long-chain fatty acids and other long-chain compounds in certain yeasts, such as Candida bombicola (Daniel et al., Biotech. Lett. 20: 1153-1156 (1998)), Torulopsis magnoliae (Gorin et al. (1961), supra), and Torulopsis gropengiesseri (Jones et al., J. Chem. Soc. Perkin 1 22: 2801-2808 (1968)). The ω and ω-1 hydroxy fatty acids can also be synthesized in plants and microbes by cytochrome P450 monooxygenase-mediated oxidation of long-chain fatty acids (Lu et al. (2010), supra; and Höfer et al., J. Exp. Bot. 59: 2347-2360 (2008)). Since microbial production of ω and ω-1 hydroxy fatty acids requires long-chain fatty acids as substrates, various chemical synthesis routes have been proposed but these also require expensive functionalized substrates and multi-step processes (Metzger et al. (2009), supra; and Villemin et al., Synthesis 3: 230-231 (1984)).
In view of the above, it is an object of the present disclosure to bioengineer microbes, such as E. coli, to produce ω-functionalized fatty acids, in particular ω-hydroxy-functionalized fatty acids. This and other objects will become apparent from the detailed description provided herein.